Elevated intraocular pressure (IOP) has long been assumed to play a causative role in glaucomatous damage to the optic nerve head (ONH). It is still unclear, however, how IOP triggers the cascade of events that lead to retinal ganglion cell death. It has been proposed that the elevated IOP can 1) exert a direct mechanical strain on the connective tissues and axons in the optic nerve head, and 2) impair the blood supply to the ONH, resulting in hypoxia of the ONH tissues and subsequent cell death. Using three-dimensional (3D) reconstructions of the ONH, and principles of biomechanical engineering, we have studied the mechanical effects of elevated IOP in glaucoma. However, the relationship between the mechanical and vascular effects of elevated IOP is still unclear. What is the role of the vasculature and ischemia in the development and progression of the disease? Is ONH blood flow related to IOP-induced deformation of the ONH connective tissues? Are the robustness of the ONH connective tissues and the vasculature the key to understanding individual susceptibility to glaucoma? To answer these questions, methods for determining the relationship between IOP-induced deformation of ONH connective tissues and ONH blood flow are needed. We propose to develop a system that constitutes the essential first step in quantifying ocular perfusion, an important risk factor for glaucomatous damage. While many additional variables will need to be considered, the ability to isolate, quantify, and model co-localized vascular and connective tissue structures within three- dimensional (3D) reconstructions of the ONH will be a powerful advance in studies of ONH susceptibility. In a preliminary test, both the ONH connective tissue and retinal vasculature were revealed by spontaneous and acquired fluorescence, respectively. The proposed system will provide simultaneous, co-localized, high- resolution, 3D reconstructions of the vascular and connective tissue structures of the ONH. We also propose to quantify and correlate the regional changes in connective tissue deformation, capillary patency, and vascular volume in contralateral eyes perfusion-fixed at acute IOPs of 10 and 45 mm Hg, respectively. Within the proposed 3D reconstructions, we can perform geometric quantification and biomechanical modeling of the coupled connective tissue and vascular systems to predict IOP-induced changes in ONH blood flow. Elevated intraocular pressure (IOP) has long been assumed to play a causative role in glaucomatous damage to the optic nerve head (ONH), but the interplay between IOP-related connective tissue deformation and ONH blood flow is unclear. We propose to develop a system that will generate simultaneous, co-localized, high- resolution, three-dimensional (3D) reconstructions of the vascular and connective tissue structures of the ONH. Using these 3D reconstructions of the ONH and principles of biomechanical engineering, we can model the mechanical and vascular effects of elevated IOP in glaucoma. [unreadable] [unreadable] [unreadable]